DNA-seq or genomic RNA region of HIV-1 reverse transcriptase, protease and integrase gene
DNA-seq or genomic RNA region of HIV-1 reverse transcriptase, protease and integrase gene
All samples are collected at University Hospital for Infectious Diseases in Zagreb as a part of the study of molecular epidemiology of HIV infection. For this purpose clinical plasma samples were collected from HIV-1 patients. HIV-1 RNA was extracted from 500 μl of patient's plasma sample by using the QIAmp Viral RNA Mini Kit (Qiagen, Hilden, Germany). HIV-1 RNA was reverse transcribed with SuperScript III First-Strand Synthesis System for RT-PCR (Invitrogen, Carlsbad, CA) and HIV-1 specific primer.Amplification of target region for each sample was done in 4 separate multiplex PCR reactions using ALLinTM Taq DNA Polymerase (highQu GmbH, UK). For this purpose we constructed 22 primer pairs that span specific region of interest. After multiplex PCR, amplicons of each sample were pooled in one tube and purified with Agencourt Ampure XP beads (Beckman Coulter, Germany). Viral DNA libraries were prepared for deep sequecing with NEBNext UltraTM II DNA Library Prep Kit for Illumina (MA, USA), according to the manufacturer's instructions.